Hydrogels consist of polymer chains forming a hydrophilic, water containing network having a wide area of application within various industrial fields, in particular within the biotechnological and pharmaceutical industry. For example, hydrogels are used to immobilize biological material such as cells for transplantation or as delivery system for e.g. pharmaceutical active ingredients or nutrients. Hydrogels may also be useful as wound dressings. Furthermore, hydrogels, in particular alginate hydrogels, is widely used as thickening agents.
A widely used polymer for the formation of hydrogels is alginate. Alginate are naturally occurring, anionic polysaccharides consisting of 1,4-linked-β-D-mannuronic acid (M) and α-L-glucuronic acid (G). (Smidsrød and Skjåk-Bræk, 1990, Trends in biotechnology, vol. 8, no. 3, pp 71-78). Commercial alginates are extracted from seaweed, such as Ascophyllum nodosum, Macrocystis pyrifera, and Laminaria hyperborea, and also to some extent Laminaria digitata, Laminaria japonica, Eclonia maxima, Lesonia negrescens and Sargassum sp. Alginates may also be prepared from some alginate producing bacteria, e.g. from some Pseudomonas species and from Azotobacter vinelandii (Smidsrød and Skjåk-Bræk, 1990, supra).
Alginates are commonly used inter alia in the food industry, e.g. as stabilizers for viscosity control, or as thickening agents. Alginates are also widely used within the pharmaceutical industry and cosmetic industry, also as stabilizers, thickening agent or disintegrant. For the various purposes, alginates being rich in either guluronic acid or mannuronic acid, respectively, are available (Mancini et al. (1999), Journal of Food Engineering 39, 369-378, WO8603781, U.S. Pat. No. 4,990,601, U.S. Pat. No. 5,639,467).
Due to alginates bio compatibility and ability to gel in presence of divalent cations such as e.g. calcium ions, alginate is also commonly used for encapsulation of cells (Nebel, R. L., Balme, J., Saacke, R. G. and Lim. F. (1985), J. Anim. Sci. 60:1631-1639, Lim, F and Sun, A. M., (1980) Science 210: 908-9100, WO 2006/106400, EP0922451, U.S. Pat. No. 6,596,310, Torre et al. (1998), S.T.P. Pharma Sciences, 8 (4), pp. 233-236, Torre et al., (2000), Biomaterials, 21, pp. 1493-1498, Torre et al. (2002), Journal of Controlled Release, 85, pp. 83-89, Faustini et al, (2004), Theriogenology, 61, 173-184, Weber et al. (2006), Journal of Biotechnology, 123, pp. 155-163.
Alginate gels are also useful for immobilising various materials. For example, WO2008/004890 describes biopolymer particles useful for preservation of spermatozoa, and wherein the biological material is embedded in a polymer particle being solid throughout the whole diameter of the particle. By embedding the spermatozoa in the alginate hydrogels instead of encapsulating the spermatozoa, leaving the spermatozoa in the fluid core of the capsules, the cells are immobilized within the alginate gel network, so restricting the motility of the cells during storage.
Alginate hydrogels, e.g. alginate gels used for encapsulation or entrapment of various materials, may be prepared by mixing a solution of the material to be entrapped with a sodium alginate solution, and adding this solution into a solution containing multivalent cations, usually divalent cations such as calcium ions (e.g. a solution of CaCl2) (Smidsrød and Skjåk-Bræk, 1990, supra).
U.S. Pat. No. 6,497,902 disclose another method for preparing biocompatible hydrogels, such as alginate hydrogels, comprising mixing the cells to be embedded, alginate salt and a calcium releasing agent, and thereafter adding a calcium releasing compound to said mixture to form a cross-linked gel. According to U.S. Pat. No. 6,497,902, the calcium releasing agent may be D-glucono-δ-lactone (GDL).
The method disclosed in U.S. Pat. No. 6,497,902 may be used to immobilize spermatozoa useful for artificial insemination (e.g. for the preparation of alginate hydrogels disclosed in WO 2008/004890). For example, diluted and cooled (4° C.) spermatozoa may be added to a solution comprising dissolved sodium alginate and suspended calcium carbonate, and optionally a cryoprotectant such as glycerol, and thereafter initiating gelling and obtaining the desired alginate hydrogel by adding a solution comprising GDL. The addition of the GDL results in the formation of gluconic acid which in turn will react with water and form H3O+. The increase in H3O+, and the presence of calcium carbonate, results in the release of CO2 and Ca2+. The providing of Ca2+ by the adding the GDL results in formation of the alginate hydrogel with embedded spermatozoa.
After gelling has occurred, the containers filled with spermatozoa embedded in alginate may be cryopreserved in liquid nitrogen, thus providing cryopreservation of spermatozoa having exceptionally long shelf life.
However, the present inventors have experience that the use of GDL in preparing alginate hydrogels according to the method disclosed in U.S. Pat. No. 6,497,902 involves several drawbacks. When preparing alginate gel comprising immobilized spermatozoa according to the method described above, it is vital that GDL is added to the solution immediately after the GDL solution is prepared to avoid spontaneous gelling. GDL must be added in dissolved form rather than as powder to avoid local areas/zones with high concentrations of gluconic acid initially, which would be detrimental to the spermatozoa. Furthermore, after the addition of GDL, a period of increasing viscosity will follow as a result of the initiation of the gelling reaction. Due to the increasing viscosity, the container used to form the hydrogel must be filled rather quickly. During the accrued time for transforming the dissolved GDL to glucuronic acid, the solution is therefore transferred to suitable containers (such as e.g. mini straw provided from IMV, L′Aigle, France) for further gelling and immobilizing of the desired biological material. The method disclosed in the prior art do therefore result in a rather short and inflexible time schedule for preparing the hydrogels comprising the desired biological material and are a substantial disadvantage from an industrial point of view.
Due to the drawbacks of the method described above, there is a need for an improved method for preparing hydrogels, in particular a method being suitable for preparing hydrogels on an industrial scale.
Various other methods for preparing hydrogels have been described in the prior art. Various reports disclose the utilization of enzymes which upon being subjected to a specific substrate provides for various reactions that in the end results in crossbinding of various types of polymers.
For example, CN 101439206 discloses inter alia the use of polymers comprising a phenolic hydroxyl unit and dioxygenase in an enzyme catalyzed process for the preparation of polymer gels.
Johnsen et al. (2010), ACS Applied Materials & Interfaces, 2(7), pp. 1963-1972, report the preparation of PEG based hydrogel being polymerised using glucose oxidase. The glucose oxidase catalyses the oxidation of β-D-glucose, and the subsequent use of oxygen to generate the flavin adenine dinucleotid enzyme cofactor, results in formation of H2O2. By combining ferrous ions with this enzymatic H2O2 production, primary hydroxyl radical species are produced that further reacts with the acrylated monomers.
The use of H2O2 and horse radish peroxidase in preparation of hydrogels have also been reported, c.f. e.g. Kurisawa et al., (2010), J. of Materials Chemistry, 20(26), pp. 5371-5375, Sakai et al, (2009), Biomaterials, 30(20), pp. 3371-3377, Sakai and Kawakami (2006), Acta Biomaterialia, 20017, 3, pp. 495-501, Lee et al. (2009), J. of Controlled Release, 134, pp. 186-193, Wang et al. (2010), Biomaterials, 31, pp. 1148-1157.
Hydrogels prepared by the use of oxidases and H2O2 are inappropriate for some application since H2O2 is a strong oxidant.
There is therefore still a need for an improved and simplified process for the preparation of alginate hydrogels, in particular hydrogels suitable for immobilizing biological material.